Yazarlar WSN layık Aziz Louis Üniversitesi Rektörlük Araştırma Fonu kabul etmek istiyorum

1.. Nüfus Kafes Bakım <ol><li> 12 saat ışık-karanlık çevrimi ile 25 ° C &#39;de nüfus kafesleri koruyun. Sürece kontrol ve deney grupları aynı aydınlatma koşullarına maruz kaldığı zaman, bu teknik, standart bir laboratuvar ortamında gerçekleştirilebilir. </li><li> Kadın elma suyu agar plakaları üzerinde gece yumurtalarını bırakmak için izin verir. </li><li> 24 saat boyunca 25 ° C &#39;de, yeni biriken yumurta plakaları koruyarak yeni yumurtadan çıkmış larva toplayın. Taranmış larva çekmek için plakanın ortasına maya küçük topak yerleştirin. </li><li> Elma suyu plakasının merkezinde maya hamur içine göç 1. instar larvaları toplamak. Taze elma suyu plaka aktarmak için metal bir spatula kullanın. Ek maya hamur tahliller yapılana kadar yeterli gıda olmadığından emin olmak için ilave edilebilir. Üzüm suyu plakaları da elma suyu levhaların yerine kullanılabilir. </li><li> Geç 2.-erken 3 instar l elde1. 40-48 saat için yaş Instars izin vererek arvae. Bu aralıkta yemleme 13 sabittir. Her larva molt bu yapıda belirgin değişiklikler olduğu gibi yaş ağız kanca incelenmesi ile teyit edilebilir. </li><li> 2. geç-erken 3. instar larvaları yavaşça ve yaygın su ile elma suyu tabak yıkama ve bir gözenekli filtre üzerine toplanmasıyla larvaları davranış testleri için toplanır. Larvalar daha sonra bir agar plakası üzerine aktarılır. </li><li> Tüm analizler kontrol ve deney hayvanları kullanarak paralel olarak gerçekleştirilir. </li></ol> 2. Davranış Paradigma - Locomotion <ol><li> Bir 100 mm doku kültürü çanağı içinde bir% 2 agar tabaka üzerinde tek bir 3. deri değiştirme safhasında larva yerleştirin ve larva 30 saniye süre ile uyum sağlamaya izin verir. Larva agar alt-tabakanın yüzeyi üzerinde vücutlarını itmek için cephalopharyngeal sklerit (ağız kanca) kullanın. Bu ayrı bir tabakta yapılır çünkü it hayvan neredeyse maya gibi aynı renkte olduğu gibi maya çözelti içinde vücut kasılmaları görselleştirmek için daha zordur. </li><li> 1 dakikalık bir süre için alt-tabaka üzerinde ön hareketine her posterior dikkat edin ve kaydedin. n = her bir genotip için 20. En fazla 10 hayvan plaka başına test edilmelidir. </li></ol> 3. Davranış Paradigma - Yemleme <ol><li> Küt paslanmaz çelik 5. cımbız kullanarak dikkatli bir şekilde% 2 aktif ekmek mayası çözeltisi, 5 ml ile kaplanmış bir agar dolu plakasının merkezine lokomotor agar plakasından 3. instar larvası aktarın. Maya zamanla razı olacak gibi çözüm homojen olduğundan emin olun. Ne maya çözeltisi içinde büyük ölçüde larva davranış gözlem kolaylaştırmak, yer ve yem kalacaktır. Ağız kanca kasılmaların hızı, doğrudan 14 yutulur gıda miktarı ile ilişkilidir. </li><li> Larva 30 saniye gelmesini bekleyin. </li><li> Gözlemlemek ve sayısını kaydetmek1 dakikalık bir süre boyunca ağız kanca kasılmalar. n = her bir genotip için 20. </li></ol> 4. Larva Gut Diseksiyonlarında <ol><li> 3-çukurlu nokta cam 1x fosfat tamponlu tuz (PBS) ve yerinde bir% 4 EM dereceli formaldehit tespit çözüm olun. </li><li> Dikkatle, 1x PBS solüsyonu kullanarak 3-iyi bir cam tabak geç 3 instar larva cesareti dolaşıp proventrikülüs değişmeden kalır emin her zaman yapmak teşrih. Bir forcep ile, posterior ucundan tutun ve diğer, ağız kanca tutun. Posterior uç hareketsiz tutarken, hafifçe cesareti erişmek için ağız kanca çekin. Ilişkili herhangi bir doku (tükrük bezleri, beyin, vücut yağ, vb) çıkarın, daha sonra formaldehit düzeltmeyi içeren 3-iyi bir çanak her gut aktarın. 3. instar larva dolaşıp çünkü gelişme bu aşamada, larva pupariation için hazırlık beslenme ateşkes kullanılan ve proventrikülüs maya temizlenir. </li> &lt;li&gt; opak bir doku kültür kutusuna bir gece boyunca 4 ° C&#39;de inkübe edin bağırsaklar. </li><li> Kuyulardan formaldehit tespitine çözüm çıkarmadan önce, mide çekumlardan kaldırmak ve projeksiyonlar açıkça engel olmadan görülebilir böylece proventriculus gelen ~ 150 mikron 2 midgut klibi. </li><li> Kuyulardan formaldehit düzeltme çıkarın ve 1x PBT (1 x PBS,% 0.1 &#39;lik bir proteaz içermeyen sığır serumu albümini,% 0.1 Triton X-100) tampon çözeltisi ile değiştirin. İyice 1x PBT 10 dakika boyunca cesaretin 6x yıkayın. Yıkar yaparken mekanik bir döndürücüde doku örnekleri yerleştirin. </li><li> 10 -6 M serotonin sinyalini geliştirmek için 5-HT 1 saat boyunca 4 ° C&#39;de inkübe edin. İyice 1x PBT 10 dakika boyunca cesaretin 6x yıkayın. Önceki çalışmalar eksojen 5-HT bu konsantrasyon immünohistokimyasal analizler nöronal mimari veya varis yoğunluğunu etkilemez gösterdi ve sadece gürültü oranı, 15-16 sinyal artırır var. </li><li> 4 inkübe &amp;# 176; C gecede, anti-serotonin primer antikor (fare ya da tavşan poliklonal büyüdü monoklonal) içinde. İyice 1x PBT 10 dakika boyunca cesaretin 6x yıkayın. Yıkar yaparken mekanik bir döndürücüde doku örnekleri yerleştirin. </li><li> (; 1:400 seyreltme Alexa Fluor 568 keçi anti-fare ya da anti-tavşan IgG), ikincil antikor olarak, 90 dakika boyunca 4 ° C&#39;de inkübe edin. İyice 1x PBT 10 dakika boyunca cesaretin 6x yıkayın. Yıkar yaparken mekanik bir döndürücüde doku örnekleri yerleştirin. </li><li> Mekanik bir karıştırıcı üzerine, 10 dakika boyunca 4 mM sodyum karbonat içinde inkübe, daha sonra n-propil gallate/20 mM sodyum karbonat% 4 montaj ve floresan altında görünüşüdür. Sodyum karbonat mountant ortam içinde kullanılan tampon ve pH örnekleri dönüştürmek için kullanılır. </li><li> Analizi için 400X büyütme ile immunohistokimyasal doku örneklerinin görüntüler yakalayın. </li></ol> 5. Sinir Devre Analizi <ol><li> Nörit lifler (nicelleştirilmiştir sayısı ve büyüklüğü varikositlerdenve dallanma derecesi) Neuroleucida ve Neuroexplorer kullanarak. Ancak, bu da elle gerçekleştirilir veya Basit Neurite Tracer (online indirilebilir ücretsiz bir uygulama) kullanılarak yapılabilir. </li><li> Proventriculus beyinden gelen tek lif projeksiyonları Trace ve varis numarası, şube ve birim uzunluk başına büyük varisler sayısını ölçmek. Beyinden çıkıntı aksonal lifler nüks sinir bindirildiğini ve onlar onlar ayrı proventrikulus ve salkımlı ulaşana kadar analiz için erişilebilir değillerdir. </li></ol>

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Biz ifşa hiçbir şey yok.

Geç embriyojenez sırasında oluşur serotonerjik stomatogastric devresi, anormal gelişimi, olgun fonksiyonunu etkiler. Nörit mimarisinde değişiklikler gut (bir maya çözelti içinde ağız kanca kasılma ile ölçülen) oranı besleme devresinin fonksiyonel çıkışı, (Şekil 1) ile ilişkili olabilir innerve. Drosophila UAS-Gal4 bipartit sisteminin kullanımı mümkün özellikle belirli bir dokuya belirli transkriptin yukarı-veya aşağı-düzenlenmiş ekspresyonunu hedeflemek için...

Drosophila nedeniyle hızlı bir nesil zaman, düşük deney maliyetinin, genetik ve çevresel faktörlerin manipüle ve kontrol yeteneği nöral devre geliştirme eğitimi için son derece güçlü bir model sistem. Nöron, nöronal yol bulma ve sinaptogenez insan ve Drosophila arasında muhafaza edilir, bu nedenle, oluşturma, bakım ve nöral devreleri değiştirerek mekanizmalar da korunmuştur. Gibi serotonin (5-hidroksitriptamin veya 5-HT) gibi klasik nörotransmiterler, olgun sinir devresinde 1-3 sinyal molekülleri olarak rollerini kabul etmeden önce, büyüme faktörleri olarak görev yapabilir Daha önceki çalışmalar, olgun nöronların 4&#39;ün bağlantı değiştirebilir embriyojenez sırasında 5-HT seviyelerini tedirgin göstermiştir. Diğer Helisoma kültürlü nöronlar için 5-HT ektopik uygulama nörit gelişimini hem de sinaptogenez 5-7 baskı altında olduğunu göstermiştir. D&#39;derosophila, gelişimsel 5-HT seviyeleri ters olarak CNS 8 foregut için çıkıntı nörit uzunluğu boyunca varis sayısı ve boyutu, hem de aborization derecesi ile ilgilidir. Serotonerjik sinir Drosophila 8-9 de dahil olmak üzere, çeşitli türlerde beslenme davranışları modüle ettiği gösterilmiştir. Drosophila besleme devresi foregut beyinden aksonal çıkıntıların gelişiminde değişiklikler ile fonksiyonel çıkış (besleme) ilişkilendirmek için bir model olarak kullanılabilir nispeten basit bir devredir. Schoofs et al. Drosophila larva besleme kasları 10 etkileyebilir merkezi desen jeneratörleri tarafından düzenlenir olduğunu göstermiştir. Spesifik kas anatomi tam olarak anlaşılamamakla birlikte, bu antennal sinir, üst çene siniri ve protorasik bezle aksesuar sinir katılan kas hedefler için sorumlu olduğu gösterilmiştirbeslenme davranışı. Omurgasız besleme kas ve sinir anatomisi ile ilgili en veri Calliphora larvaları ile sınırlıdır. İkinci instar larvalarının besleme oranı cephalopharyngeal sklerit (ağız kancalar) geri çekilmesi ile değerlendirilmiş ve tekrarlanabilir ve yüksek verimli edilebilir. Cephalopharyngeal plakaları frontal sinir yoluyla santral 5-HT nöronların lifleri tarafından innerve edilir. Proventrikülüs veya foregut, midgut salkımlı ve (Şekil 1) 11-12 foregut kasılmaları sorumludur serotonerjik lifler (sinir nüks) tarafından innerve edilir. Aksonal dallanma değişiklikler ve nörit uzunluğu boyunca varikositlerden sayısı ve büyüklüğü, immünohistokimyasal teknikler kullanılarak kantifiye edilebilir. Ya doğrudan ya da dolaylı olarak, geliştirme sırasında nöronal 5-HT idare etme şekillendirmeler değişiklikler ile değerlendirilir ve korele edilebilir, bu besleme devresinin fonksiyonel çıkışı değiştirebilirnörit mimari gy.

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			<td height="40" style="height:40px;width:217px;">Eclipse E-800 Microscope</td>
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			<td style="width:251px;">Used to analyze gut fiber architecture, not necessary to have</td>
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			<td height="60" style="height:60px;width:217px;">G-2E/C TRITC EX 528-553</td>
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			<td style="width:115px;">MRH00400</td>
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functional analysis of the larval feeding circuit in drosophila

Drosophila melanogaster larvalarda serotonerjik besleme devresi devrenin geliştirilmesi sırasında kritik önem nöronal substratları incelemek için kullanılabilir. Devrenin işlevsel bir çıkış kullanarak, besleme, stomatogastric sisteminin nöronal mimarisinde değişiklikler görülebilir. Besleme davranış beyin sinirlerini almak ağız kanca, retraksiyonun hızını gözlemleyerek tarafından kaydedilebilir. Larvalar bir ağar tabakayı karşısında çapraz onların ağız kanca kullanmak beri lokomotor davranışlar, beslenmesi için bir fizyolojik kontrol olarak kullanılmıştır. Besleme davranış değişiklikleri neurites aksonal mimarisi ile ilişkili olabilir gut innerve. Immünohistokimya kullanılarak bu değişiklikler görselleştirmek ve ölçmek mümkündür. Onlar manipülasyonlara çok hassas olarak davranış paradigmaları sırasında larvaların Yanlış kullanım verilerini değiştirebilir. Nörit mimarlık inervasyonu Uygun görüntülemegut kesin varikositlerden sayısı ve büyüklüğü ölçülmesi hem de dal düğümlerin ölçüde için çok önemlidir. En devrelerin analizi sadece nörit mimari veya davranışsal etkilerinin görselleştirme için izin, ancak bu model bir nöronal mimari engelli devrenin işlevsel çıktı korelasyon sağlar.

Drosophila larva serotonerjik besleme devresi sinir sistemi gelişiminde belirli faktörlerin etkisini gözlemlemek için son derece etkili bir model olarak hizmet edebilir. Besleme hızı ile ölçülmesi, bu fonksiyonel çıkış (Şekil 1) ile birlikte besleme devresinin aksonal mimarisi bağlamak mümkündür. Larvalar agar yüzeyi boyunca kendilerini itmek için ağız kanca kullandığından lokomotor deney, ağız kancaların geri çekme için fizyolojik bir kontrol olarak kullanılır. ...

Watch this Scientific Journal Video about Functional Analysis of the Larval Feeding Circuit in Drosophila at JoVE.com

Functional Analysis of the Larval Feeding Circuit in Drosophila

Drosophila melanogaster larvaları besleme devresi besleme hızındaki değişimler stomatogastric sinir devresi değişiklikler ile korelasyon sağlayan bir basit ama güçlü bir model oluşturuyor. Bu devre, ağız kanca projeksiyonlar hem de foregut göndermek merkezi serotonerjik nöronların oluşmaktadır.

Içinde larvaların Devre Fonksiyonel Analiz Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

functional-analysis-of-the-larval-feeding-circuit-in-drosophila

Research

JoVE Journal

Neuroscience

Functional Analysis of the Larval Feeding Circuit in Drosophila

10.2K Görüntüleme.  Saint Louis University School of Medicine.  Drosophila melanogaster larvaları besleme devresi besleme hızındaki değişimler stomatogastric sinir devresi değişiklikler ile korelasyon sağlayan bir basit ama güçlü bir model oluşturuyor. Bu devre, ağız kanca projeksiyonlar hem de foregut göndermek merkezi serotonerjik nöronların oluşmaktadır. 

Video: Functional Analysis of the Larval Feeding Circuit in Drosophila

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3 Larvaların segmental sinirler Görselleştirme Rd Instar Drosophila Larvaların Hazırlıkları

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Nörobilim

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Drosophila Larvaların segmental Aksonlar içinde Sinaptik veziküller in vivo Görselleştirme

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Morfolojik analiz Drosophila</emGenetik Mozaikler> Larvaların Periferik Duyusal Nöron Dendritler ve Aksonlar

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Içinde proprioseptörlere Görselleştirmesi<em&gt; Drosophila</em&gt; Larva ve Pupa

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Larva, Pupa ve Erişkin diseksiyonu ve İmmünohistokimya<em&gt; Drosophila</em&gt; Retinaları

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

In Koku Reseptör Nöronlar Tüm Dağı ile immün<em&gt; Drosophila</em&gt; Anten

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Canlı Görüntüleme<em&gt; Drosophila</em&gt; Larva nöroblastomun

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

En fenotip Karakterizasyonu: Niceleme Matters Neden<em&gt; Drosophila</em&gt; Larva Nöromüsküler Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Kaldırılması<em&gt; Drosophila</emDuyu Nöronlar ve Epidermal Hücre İmmünofloresan Analizi Larvaların Fileto dan&gt; Kas Dokusu

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila larva nöromüsküler kavşak sinaptik akımlardan Fokal Macropatch kayıtları

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...